1. Field of the Invention
The present invention relates to gene amplification and, more specifically, to a method for the linear amplification of small polynucleotides, such as microRNAs.
2. Description of the Related Art
The central dogma of molecular biology and genetics is that within a cell DNA is transcribed into messenger RNA (mRNA) and mRNA is subsequently translated into a corresponding protein. As with many general biological principles, this view appears to be generally valid. Recent evidence, however, including sequencing of the human genome and the continued sequencing of relevant animal models has demonstrated that there appears to be more information in the genome in terms of protein synthesis control than previously realized. Multiple lines of research are beginning to demonstrate that non-protein coding RNAs (ncRNAs) may play a central role in translational regulation. For example, <3% of mRNAs are translated into proteins. Importantly, microRNAs (miRNAs) are a short 17-25-nucleotide (nt) class of RNA molecules that have been shown to have critical functions in a wide variety of biological processes. miRNAs are synthesized from larger miRNA transcripts that fold to produce hairpin structures and serve as substrates for an enzyme of the RNase III family termed cytoplasmic Dicer. They are endogenous noncoding RNAs, which can regulate gene expression of multiple mRNAs. Since the identification of miRNAs in 1993, a growing list of additional miRNAs has been expanding steadily. Approximately 300-400 miRNAs have been identified in human, and each of these miRNAs has been estimated to interact with 5-10 mRNAs. It is estimated that approximately 30% of all expressed genes are regulated by miRNAs. With the exception of certain viruses, most miRNAs reduce gene expression through reduction of the abundance and/or the translational efficiency of target mRNAs.
Studies have shown that miRNAs can be expressed in a tissue-specific or cell-specific manner. Essentially, a mosaic of miRNA expression levels regulates cell growth, differentiation, and other critical parameters throughout various cell types and tissues. miRNA expression levels are controlled tightly during development and are critical for generalized homeostasis. Furthermore, many pathological conditions appear to be related to dysfunction of miRNA expression, including several types of cancer, although this work is in its infancy. miRNA investigation is also being initiated within neurons and dendrites, and it is postulated that miRNA dysfunction has a profound impact on the pathophysiology of neurodegenerative disorders, including Fragile X syndrome, Tourette syndrome, polyglutamine repeat disorders, as well as Alzheimer's disease (AD) and related dementing illness.
miRNA expression profiling is an ideal method to study expression levels and regulation of individual miRNAs, similar to current paradigms examining mRNA expression levels at the global, regional, and cellular level. However, miRNA profiling poses more challenges than mRNA profiling. One major obstacle when profiling miRNAs is the low expression level. miRNAs are estimated to constitute only 0.001% of total RNA. As a result, large quantities of input RNA are required for miRNA profiling. This requirement makes it difficult to study tissue-specific and cell-type specific expression of miRNAs. Another difficulty of profiling miRNAs is their small size, as they are predominantly 22-25 nt. The small size of miRNAs makes direct amplification difficult with conventional RNA amplification and/or qPCR-based strategies, although several groups have performed modification procedures of RNA amplification methods to identify individual miRNAs in brain. Unlike mRNAs, miRNAs lack an appropriate sequence to anchor a primer for the first strand DNA synthesis, which is typically the first step of the majority of RNA amplification procedures. Moreover, the small size of miRNAs makes them difficult to hybridize to array platforms, because of the unstable hybridization of short oligonucleotides to the complementary sequences. Unfortunately, positive signals generated by short oligonucleotides are generally weak, which reduces the overall detection sensitivity of the assay, and high background is typically a problem due to high concentrations of probe and longer exposure times required for signal detection.
Several methods are currently attempted to amplify miRNAs. In general, these methods attempt to attach a known sequence to target miRNAs through ligation or terminal transferase, which is followed by amplification step through either DNA or RNA synthesis. However, these methods may increase the signal strength but do not substantially increase the sensitivity of the method, because they invariably increase the background nonspecific hybridization. Several methods employ direct labeling of total RNA (presumably including miRNAs) followed by hybridization to immobilized probes. Direct labeling methods suffer from low sensitivity and specificity, and typically require starting material>10-20 μg. To increase the sensitivity and specificity, small RNAs can be enriched via acrylamide gel electrophoresis. The enrichment of small RNAs increases the specificity, but not the sensitivity of miRNA profiling due to the low yield of miRNAs. This method is suitable for Northern blot analysis of individual miRNAs, but may not be sensitive enough for profiling of multiple miRNAs from a small input source. Alternative methods have been employed to increase the signal strength of miRNA species for greater detection capabilities. These approaches include the use of modified nucleic acids, such as locked nucleic acids, to increase the affinity and stability of hybridization to specific probes or using tail labeling methods to increase the number of labeled nucleic acids within miRNA species. Such methods are viable for large quantities of input RNA, but have somewhat limited benefits with small samples due to the relatively nonspecific nature of locked nucleic acid and tail end labeling. Another method to amplify miRNA employs real-time quantitative PCR (qPCR). This method has been demonstrated to be successful for individual miRNAs when sample RNA is abundant. However, qPCR miRNA profiling is difficult to ‘scale up’ for minute quantities of input RNA species, since each miRNA has to be amplified and detected separately. Moreover, the method is difficult to perform with multiple samples and therefore suffers low throughput as compared to medium- and high-throughput array methods. Thus, there is a need for improved miRNA amplification methods that overcome drawbacks in existing methods.